The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR-TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR-TS is essential for parasite survival and represents a promising target for drug discovery.